Infection of the central nervous system (CNS) results in HIV-associated neurological disorders (HAND) in up to 50% of ART-treated individuals. Infection of microglia in the CNS is involved in the etiology of HAND, as microglia express both CD4 and CCR5 on their surface and are infected by HIV-1 in vivo. In addition to involvement in HAND, HIV-1 infection of microglia generates a reservoir of replication-competent virus that persists in ART-treated individuals. Knowledge about mechanisms underlying HAND and HIV-1 latency in microglia is limited, due in considerable part to the absence of tractable and robust model systems that can investigate HIV-1 infection of this important cell type. However, it has recently become possible to generate induced microglia (iMicroglia) from induced pluripotent stem cell lines. Importantly, iMicroglia possess functional properties similar to microglia in vivo, including their RNA expression profile, secretion of cytokines in response to inflammatory stimuli, and phagocytosis of CNS substrate. The research proposed in this exploratory R21 will develop a new model system? iMicroglia ? to investigate mechanisms underlying HAND and latent infection of the CNS. The research involves two Specific Aims. Aim 1 will use high-field NMR and single cell RNAseq (ssRNAseq) to identify metabolic and genetic pathways that are perturbed by HIV-1 infection. Aim 2 will utilize a dual-fluorescence HIV-1 reporter virus and cell sorting to obtain infected iMicroglia that harbor latent integrated viruses or are permissive for viral replication. SsRNAseq will identify differentially expressed genes that are associated with viral replication or latency; latency reversal agents will be evaluated and compared to their activities in CD4+ T cells. Completion of the proposed research may serve as the foundation for novel therapeutic approaches, as it will provide mechanistic insight into HAND and HIV-1 latency.